PROJECT SUMMARY/ABSTRACT Tumor resistance to radiotherapy remains a critical barrier to improving outcomes for patients diagnosed with locally advanced, unresectable cancers. Cellular sensitivity to ionizing radiation (IR) is governed by intracellular and extracellular factors. To overcome tumor radioresistance, drugs that sensitize tumor cells to ionizing radiation (IR) are used. Non-targeted cytotoxic chemotherapies given concurrently with radiotherapy have demonstrated improved tumor control and overall survival in cancer patients. However, since this paradigm shifting approach occurred in the 1980's there has been a shocking lack of progress in developing molecularly targeted radiosensitization approaches. Improving the therapeutic ratio of IR in combination with systemically delivered drugs can be achieved by two general approaches, 1) using drugs that block DNA damage repair responses 2) delivering radiosensitizing drugs selectively to tumors. These two methods are orthogonal techniques to achieve the same end goal of increasing IR induced tumor kill while reducing normal tissue toxicities. To address both of these fundamental cancer therapy problems, we have focused on target discovery and tumor selective drug delivery vehicles. With regards to target discovery, our recent studies on non-canonical CRAF functions led to our discovery of an unexpected role for PAK in DNA damage repair and sensitivity to IR. PAK is comprised of a family of six kinases subdivided in Group I and II. Importantly, PAK are involved in process central to oncogenesis, tumor aggressiveness and patient survival. To tackle tumor selective drug delivery, we are developing drug conjugated activatable cell penetrating peptides (ACPP) to selectively deliver potent radiosensitizers to tumors based on extracellular tumor protease activity. ACPP consist of a drug conjugated polycationic cell penetrating peptide and an autoinhibitory polyanionic peptide separated from each other by a flexible peptide linker. This peptide linker is specifically cleaved by proteases enriched in the extracellular tumor microenvironment. While ACPP is intact, the drug conjugated cell penetrating peptide is neutralized (i.e. held in a ?pro-drug? state) by the polyanionic peptide so that the drug cannot gain access to its intracellular target. Tumor microenvironment proteases cleave ACPP and release the drug conjugated cell penetrating peptide, which is then taken up by tumor cells. The goals of our proposal are to gain insight into how PAK governs radioresistance and then therapeutically exploit this with targeted PAK inhibitors. In Aim 1, we will genetically determine the mechanisms through which Group I and II PAKs govern IR resistance. In Aim 2, we will pharmacologically test the ability to radiosensitize tumors with small molecule PAK inhibitors. In Aim 3, we will test if tumor targeted ACPP increase the therapeutic ratio of conjugated PAK inhibitors. Our approach has complementary innovations in both DNA damage target discovery and tumor selective drug delivery. Combining these approaches will lay a foundation for moving away from non-targeted cytotoxic radiosensitization to biomarker driven molecularly guided radiosensitization.